T cell immunodominance epitope peptide of SARS-CoV-2 nucleocapsid protein mutation site and application of T cell immunodominance epitope peptide

Abstract

The invention provides a T cell immunodominance epitope peptide of an SARS-CoV-2 nucleocapsid protein mutation site and application of the T cell immunodominance epitope peptide, and belongs to the technical field of biological medicine. According to the invention, a group of dominant epitope peptide compounds capable of specifically targeting mutation sites and activating T cell response are developed by screening and identifying dominant epitopes aiming at N protein of an Omicron mutant strain JN.1 substrain. The epitope peptide can induce an organism to generate an effective specific T cell immune response aiming at a current main epidemic Omicron pedigree JN.1 substrain mutation site, so that the cellular immunocompetence of the organism is enhanced. A novel coronavirus vaccine, a medicine or an antigen detection kit developed based on the epitope peptide compound can provide possibility for effectively removing virus infected cells, reducing continuous existence of viruses in vivo and identifying new coronavirus infection, so that SARS-CoV-2 breakthrough infection is diagnosed and prevented in an early stage, and the long-term health burden of a patient is relieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a T cell immunodominant epitope peptide at a mutation site of a SARS-CoV-2 nucleocapsid protein and an application thereof. Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the Sarbecovirus subgenus of the β-coronavirus genus and is the seventh known coronavirus that can infect humans. The incubation period of this virus infection is usually 1 to 14 days, and the typical imaging feature after infection is ground-glass lesions on chest computed tomography (CT). From a clinical perspective, the main symptoms of SARS-CoV-2 infection include fever, dry cough, shortness of breath, headache, and pneumonia. Depending on the severity of the disease, the clinical classification of COVID-19 can be divided into mild, common, severe, and critical. In addition, COVID-19 is a multi-system debilitating disease that may last for several years or even lifelong. Its main manifestations include heart disease, diabetes, chronic fatigue syndrome, and autonomic nervous system disorders.

[0003] Among the many measures taken to prevent and control the virus, vaccines have played a key role, significantly curbing the spread of SARS-CoV-2 and significantly reducing the incidence and severity of infection. However, SARS-CoV-2 continues to evolve, leading to the emergence of variants. These variants have also gradually acquired the ability to evade immune responses, making breakthrough infections common.

[0004] The body's adaptive immune response includes two aspects: humoral immunity and cellular immunity. Humoral immunity mainly prevents the virus from entering cells by producing neutralizing antibodies, while cellular immunity clears virus-infected cells by activating T cells. Unfortunately, SARS-CoV-2 mutants have evolved many targets that can effectively escape the action of neutralizing antibodies. In contrast, cellular immunity is less affected by viral mutations, suggesting that viral antigen-specific memory T cells may play a key role in preventing severe illness.

[0005] Focusing further on the genomic characteristics of the SARS-CoV-2 virus, its genome is 30kb long and can encode at least 29 proteins. Among them, the nucleocapsid protein (N) plays an important role in the viral replication process. It is involved in the packaging of the genome and resists the antiviral activity mediated by host cell RNAse by binding to viral RNA. During viral infection, the N protein is produced in extremely large quantities. It is one of the proteins with the largest copy number and is highly immunogenic. Based on the above characteristics, vaccine design targeting the N protein is expected to provide an important direction for the development of broad-spectrum and long-lasting vaccines. However, the mainstream vaccine design at this stage mainly focuses on the S antigen that can produce neutralizing antibodies. As mentioned earlier, the SARS-CoV-2 virus variant acquires the ability to escape neutralizing antibodies through epitope mutations of the S antigen. The applicant's previous research results have shown that compared with the S antigen, the highly conserved N protein also has the ability to stimulate strong specific T cell responses (Ning J, Wang Q, Chen Y, He T, Zhang F, Chen X, et al. Immunodominant SARS-CoV-2-specific CD4 + and CD8 + T-cell responses were elicited by inactivated vaccines in healthy adults. J Med Virol 2023; 95(4): e28743.). Therefore, the problem of insufficient design of N antigen vaccines in the existing technology needs to be urgently addressed.

[0006] At present, the main SARS-CoV-2 mutants prevalent in the world are the Omicron lineage and its sublineages. Because Omicron has the largest number of mutation sites and has high transmissibility and immune escape capabilities, the time interval between its designation as a "variant under monitoring" (VUMs) and a "variant of concern" (VOCs) is only two days. In the past two years, Omicron has continued to mutate into new sub-mutants, including BA.2.75, BA.4 / 5, BF.7, XBB, BA.2.86 and JN.1. As of now, JN.1 is still the absolutely dominant prevalent variant worldwide. However, it is not clear whether the memory T cells triggered by early breakthrough infections have the ability to recognize the N protein of the currently prevalent JN.1.

[0007] In the field of immunology, specific regions of antigens - epitopes (also called antigenic determinants) are key structures for immune recognition. They include linear sequences or three-dimensional conformational structures that can specifically bind to receptors on the surface of antibodies or immune cells (such as T cell receptors TCR; B cell receptors BCR). Epitopes are usually composed of amino acid residues, polysaccharide residues or nucleotides, and are the basic units for activating immune responses. For immune responses mediated by T cells, antigens will be extracted and degraded by antigen-presenting cells (APCs). The short peptide fragments subsequently formed will bind to major histocompatibility complex (MHC) molecules and be presented to the cell surface. These short peptide fragments are called T cell epitopes. Depending on the type of binding to the MHC molecule, T cell epitopes are divided into two types: one is a short peptide that binds to the MHC class I molecule, usually containing 8 to 12 amino acid residues. This epitope will be recognized by CD8 + T cell recognition; the other is a short peptide that binds to MHC class II molecules, generally containing 12-18 amino acid residues, which will be recognized by CD4 + T cell recognition.

[0008] In an immune response, not all epitopes can effectively activate the immune system. Only dominant epitopes that have been immune-selected can induce the body to produce specific and efficient T cell or B cell responses. The identification of dominant epitopes is of great value for understanding disease mechanisms, developing immune detection methods, designing diagnostic reagents, and developing epitope vaccines. Clarifying the T cell immunodominant antigen epitopes and their variation characteristics is extremely important for the precise design of vaccine antigen components. This not only enables new vaccines to more effectively adapt to the mutation trends of the virus, but also significantly enhances the breadth and depth of vaccine-induced immune protection. However, in the context of breakthrough infection, the immunodominant epitope spectrum of the N protein of the JN.1 strain is still unclear.

[0009] In view of the frequent breakthrough infections caused by the currently prevalent SARS-CoV-2 mutant strains, the existing vaccines are no longer able to provide effective protection, and there is an urgent need to optimize and upgrade the vaccines. The present invention provides a new T cell epitope, which aims to provide potential candidate molecules for the development of new vaccines against SARS-CoV-2 mutant strains, thereby promoting the development of broad-spectrum vaccines against N antigens, so as to more effectively respond to the practical challenges brought about by the frequent breakthrough infections. At the same time, the T cell epitopes provided by the present invention also have the potential for application in new coronavirus drugs and diagnostic reagents, and can provide more powerful support and protection for antiviral infections. Summary of the Invention

[0010] In view of this, the purpose of the present invention is to provide a T cell immunodominant epitope peptide targeting the mutation site of the SARS-CoV-2 nucleocapsid protein and its application. By using in vitro stimulation experiments, people who developed breakthrough infection after vaccination with the inactivated SARS-CoV-2 vaccine were used as research subjects to detect the memory T cell response signal levels and characteristics against the N protein of the Omicron mutant strain JN.1 substrain. Screening work was carried out around the dominant epitope spectrum of the N protein to explore the immunodominant epitopes targeting the SARS-CoV-2 mutation site, providing potential candidate molecules and R&D strategies for the optimization and design of a broad-spectrum SARS-CoV-2 vaccine.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides a T cell immunodominant epitope peptide at a mutation site of a SARS-CoV-2 nucleocapsid protein, wherein the amino acid sequence of the T cell immunodominant epitope peptide is shown as SEQ ID NO.2 or SEQ ID NOs.4-5 or SEQ ID NOs.10-12 or SEQ ID NO.14.

[0013] The present invention also provides a nucleic acid molecule encoding a T cell immunodominant epitope peptide at the mutation site of the SARS-CoV-2 nucleocapsid protein.

[0014] The present invention also provides a T cell immunodominant epitope peptide complex at a SARS-CoV-2 nucleocapsid protein mutation site, comprising the T cell immunodominant epitope peptide.

[0015] The present invention also provides the use of the T cell immunodominant epitope peptide, the nucleic acid molecule or the T cell immunodominant epitope peptide complex in the preparation of a new coronavirus drug.

[0016] The present invention also provides the use of the T cell immunodominant epitope peptide, the nucleic acid molecule or the T cell immunodominant epitope peptide complex in the preparation of a new coronavirus vaccine.

[0017] The present invention also provides the use of the T cell immunodominant epitope peptide, the nucleic acid molecule or the T cell immunodominant epitope peptide complex in the detection of novel coronavirus antigens.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] By screening and identifying dominant epitopes in the N protein of the Omicron mutant JN.1 substrain, the present invention successfully developed a dominant epitope peptide that specifically targets the mutation site and effectively activates T cell responses. This epitope peptide can induce a broad-spectrum and highly effective cellular immune response against the Omicron mutant, thereby preventing breakthrough infection with SARS-CoV-2. Furthermore, the dominant epitope peptide described in the present invention can also be used in the development of novel coronavirus drugs and detection reagents, providing more comprehensive and powerful support and protection against novel coronavirus infection.

[0020] The dominant epitope peptide of the present invention activates the specific T cell immune response against the mutation site of the Omicron mutant strain JN.1 substrain, prompting it to secrete high levels of antiviral cytokines, thereby enhancing the body's cellular immunity, providing the possibility of effectively clearing virus-infected cells and reducing the persistent presence of the virus in the body, thereby reducing the risk of "long COVID-19" and alleviating the long-term health burden of patients.

[0021] The dominant epitope peptide targeting the mutation site of the JN.1 mutant strain provided by the present invention can improve the breadth spectrum and effectiveness of the vaccine, and provide important technical support for the development of a new generation of SARS-CoV-2 vaccines and therapeutic drugs targeting mutant strains.

[0022] The epitope peptides provided by the present invention contain no toxic components or allergens, effectively reducing the risks associated with the use of vaccines and drugs based on these epitope peptides. These vaccines, including protein subunit vaccines and nucleic acid vaccines, offer significant advantages in safety and stability, providing a more reliable guarantee for their clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the gate logic diagram used when using flow cytometry to detect intracellular factors;

[0024] Figure 2 It is a CD4 specific antigen against the N protein of the original strain Wuhan-Hu-1 and the JN.1 variant in people who developed breakthrough infection after receiving three doses of inactivated vaccine. + and CD8 + Figures showing T cell response results (A is a schematic diagram of the experimental flow chart, B and E are flow cytometry plots of representative individuals, C and F are statistical results of 57 specimens, D and G are multifunctional analyses of T cells secreting three cytokines: IFN-γ, TNF-α, and IL-2, and H is a comparison of the levels of five cytokines: IFN-γ, TNF-α, IL-2, IL-4, and IL-10 secreted by JN.1 variant N protein-specific T cells);

[0025] Figure 3It is a specific CD4 peptide library targeting the N protein mutation site and the original site in people who developed breakthrough infection after receiving three doses of inactivated vaccine. + and CD8 + Figure 1 shows the results of T cell responses (A is a schematic diagram of the experimental process, B and D are analysis of the expression levels of three cytokines, IFN-γ, TNF-α, and IL-2, in 57 specimens, and C and E are correlation analyses of T cell responses at the mutation site and those at the original site);

[0026] Figure 4 It is a specific protocol for preparing a three-dimensional matrix peptide library;

[0027] Figure 5 The three-dimensional matrix peptide library was used to screen and identify CD4 specific for the JN.1 mutant N protein. + T cell immunodominant epitopes (A shows the flow cytometry results of a representative individual, and B shows the frequency statistics of the dominant epitopes of 57 specimens screened using this method);

[0028] Figure 6 The three-dimensional matrix peptide library was used to screen and identify CD8 specific for the JN.1 mutant N protein. + T cell immunodominant epitopes (A shows the flow cytometry results of a representative individual, and B shows the frequency statistics of the dominant epitopes of 57 specimens screened using this method);

[0029] Figure 7 This is a graph showing the conservation results of the N protein amino acid sequences of the original strain Wuhan-Hu-1 and six Omicron subvariants (BA.2.75, XBB, BA.5, BF.7, BA.2.86, and JN.1) (the specific locations of the dominant epitope peptides containing the mutation sites screened are circled in the figure);

[0030] Figure 8 The diagram shows the prediction results of toxicity and allergenicity of the dominant epitope peptides containing mutation sites screened using the bioinformatics tools ToxinPred and AllerTOP v2.1, respectively. DETAILED DESCRIPTION

[0031] The present invention provides a T cell immunodominant epitope peptide at a mutation site of a SARS-CoV-2 nucleocapsid protein, wherein the amino acid sequence of the T cell immunodominant epitope peptide is shown as SEQ ID NO.2 or SEQ ID NOs.4-5 or SEQ ID NOs.10-12 or SEQ ID NO.14.

[0032] In the present invention, the SEQ ID NO. 2 is specifically QNQRNALRITFGGPSDST;

[0033] The SEQ ID NO. 4 is specifically GPSDSTGSNQNGGAR;

[0034] The SEQ ID NO.5 is specifically GSNQNGGARSKQRRP;

[0035] The SEQ ID NO.10 is specifically AALALLLLDRLNKLESKM;

[0036] The SEQ ID NO.11 is specifically LLDRLNKLESKMSGKGQQ;

[0037] The SEQ ID NO. 12 is specifically KLESKMSGKGQQQQGQTV;

[0038] The SEQ ID NO. 14 is specifically FSKQLQQSMSRADSTQA.

[0039] The present invention also provides a nucleic acid molecule encoding a T cell immunodominant epitope peptide at the mutation site of the SARS-CoV-2 nucleocapsid protein.

[0040] The present invention also provides a T cell immunodominant epitope peptide complex at a SARS-CoV-2 nucleocapsid protein mutation site, comprising the T cell immunodominant epitope peptide.

[0041] The present invention also provides the use of the T cell immunodominant epitope peptide, the nucleic acid molecule or the T cell immunodominant epitope peptide complex in the preparation of a new coronavirus drug.

[0042] The present invention also provides the use of the T cell immunodominant epitope peptide, the nucleic acid molecule or the T cell immunodominant epitope peptide complex in the preparation of a new coronavirus vaccine.

[0043] The present invention also provides the use of the T cell immunodominant epitope peptide, the nucleic acid molecule or the T cell immunodominant epitope peptide complex in the detection of novel coronavirus antigens.

[0044] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Materials used in the present invention:

[0046] 1. Research subjects: People with breakthrough infection after inactivated vaccine A total of 57 healthy adults aged 18-60 years who underwent physical examinations in this unit from December 2022 to November 2023 were recruited. Inclusion criteria: Healthy adults aged 18-60 years who have completed 3 doses of inactivated vaccine immunization and have at least one history of positive SARS-CoV-2 nucleic acid test. Exclusion criteria: Minors under the age of 18 and elderly people over the age of 60; patients with autoimmune diseases, heart failure (heart failure grade II or above), abnormal liver and kidney function, viral colds, blood system diseases, malignant tumors or other terminal diseases; women who are pregnant or breastfeeding; and those with drug abuse or alcohol dependence. This experiment was reviewed and approved by the Medical Ethics Committee of the Eighth Affiliated Hospital of Sun Yat-sen University (approval number: 2021-005-04).

[0047] 2. Synthesis of N protein peptide library: Shanghai Qiangyao Company was commissioned to synthesize it, dissolved in dimethyl sulfoxide (DMSO) to a storage concentration of 20mM, and stored in a -80℃ low-temperature refrigerator after packaging.

[0048] 3. Preparation of complete culture medium: Measure 450 mL of incomplete 1640 culture medium, 50 mL of fetal bovine serum (FBS), and 5 mL of 100× penicillin-streptomycin, mix thoroughly, and store in a refrigerator at 4°C.

[0049] 4. 1× Zombie UVTM dye: Equilibrate the reagent to room temperature and add Zombie UV TM Add 100 μL of DMSO to the solution and mix thoroughly until completely dissolved to form a 1000× stock solution. Aliquot and store in a -20°C refrigerator protected from light. When ready to use, remove the stock solution and dissolve it in phosphate buffered saline (PBS) to prepare a 1× working solution.

[0050] 5. 1× Permeabilization Buffer: Dilute 10× intracellular staining permeabilization buffer with deionized water (ddH2O) at a ratio of 1:10 to prepare 1× working solution.

[0051] 6. RPMI-1640 culture medium, 100× penicillin-streptomycin, and PBS: ThermoFisher, USA.

[0052] 7. FBS: Ecosine Biotech Co., Ltd.

[0053] 8. DMSO: Sigma, USA.

[0054] 9. Lymphocyte separation fluid (Ficoll): Haoyang Huake Biotechnology Co., Ltd.

[0055] 10. Protein transport inhibitors (including monensin): BD Company, USA.

[0056] 11. Fixation buffer, 10× intracellular staining permeabilization buffer, and Zombie UV™ Fixable Viability Kit: BioLegend, USA.

[0057] 12. Normal saline: Zhejiang Tianrui Pharmaceutical Co., Ltd.

[0058] 13. Flow cytometry antibodies (CD3, CD4, CD8, IFN-γ, TNF-α, IL-2, IL-4 and IL-10): BioLegend, USA.

[0059] Example 1

[0060] Design, synthesis and peptide library preparation of 18 amino acid overlapping peptides of N protein

[0061] 1. The SARS-CoV-2 nucleocapsid protein (N protein) has a high degree of sequence conservation among different mutant strains. Aiming at the N protein sequence of the original strain Wuhan-Hu-1, starting from the first amino acid, 18mer overlapping peptides covering the full length of the N protein were designed and synthesized (commissioned to Shanghai Qiangyao Company for synthesis). Each group contains 68 peptide segments, and the purity of each peptide segment exceeds 90%. For the sequence information of the overlapping short peptides of the SARS-CoV-2 nucleocapsid protein of the original strain Wuhan-Hu-1, please refer to CN 116284267A. Wuhan-Hu-1 N protein amino acid sequence information (419 amino acids, NCBI accession number: YP_009724397.2):

[0062] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNT

[0063] ASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDG

[0064] KMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGT

[0065] RNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTP

[0066] GSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVT

[0067] KKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGT

[0068] DYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNF

[0069] KDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAAADLDDFSKQLQQSMSSADSTQA (SEQ ID NO. 15).

[0070] 2. Analysis and comparison of the amino acid sequences of Wuhan-Hu-1 and JN.1 revealed that JN.1 had eight amino acid mutations compared to the original strain. Peptides encompassing all mutations were resynthesized, with each peptide possessing a purity exceeding 90%. Compared to the 68 peptides in Wuhan-Hu-1, JN.1 harbored 14 mutations. The amino acid information for the peptides encompassing all mutations is shown in Table 1. The remaining 54 peptides were identical to those in Wuhan-Hu-1.

[0071] JN.1N protein amino acid sequence information (416 amino acids, NCBI accession number: XPD16435.1): MSDNGPQNQRNALRITFGGPSDSTGSNQNGGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSR NSSRNSTPGSSKRTSPARMAGNGGDAALALLLLDRLNKLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQF APSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAAADLDDFSKQLQQSMSRADSTQA(SEQ ID NO.16).

[0072] Table 1 Amino acid sequence information of mutant epitopes in the N protein of the JN.1 mutant strain

[0073]

[0074]

[0075] 3. Each peptide was dissolved in DMSO to a storage concentration of 20 mM, and then 10 μL was taken out from each overlapping peptide library to prepare 68 total peptide libraries of Wuhan-Hu-1 and JN.1 N protein, respectively.

[0076] Example 2

[0077] Detection and analysis of N protein-specific T cell response levels

[0078] 1. Isolation of PBMC: Collect venous whole blood samples (4 mL) from volunteers and mix and dilute them with normal saline in equal proportions. Take the mixed diluted blood and slowly add it to a 15 mL centrifuge tube pre-filled with an equal volume of Ficoll. Be careful to keep the interface between the separation liquid and the diluted blood sample stable. Place the centrifuge tube in the centrifuge and set the centrifugation parameters to slow acceleration and deceleration, a centrifugal force of 800 g, and a centrifugation time of 20 minutes. After centrifugation, use a bus tube to discard the top layer of plasma, and then use a new bus tube to transfer the mononuclear cell layer to a new 15 mL centrifuge tube and wash it with 5 times the volume of normal saline, repeating twice. Finally, centrifuge at a centrifugal force of 250 g for 10 minutes, collect the PBMC precipitate, and freeze it in liquid nitrogen.

[0079] 2. In vitro stimulation experiment of peptide library: PBMCs were revived at 1×10 6 Cells were seeded at a density of 10 viable cells / well in a 96-well U-bottom culture plate. Subsequently, 0.15 μL of a protein transport inhibitor was added to each well, along with a total peptide pool of 68 peptides from the N protein of the Wuhan-Hu-1 and JN.1 mutant strains, to a final concentration of 1.5 μM. DMSO-stimulated wells served as negative controls. After mixing, the plates were incubated in a 37°C incubator for 5 hours to stimulate the cells.

[0080] 3. ICS experiment:

[0081] (1) Cell collection and live-death cell staining: After stimulation with the 68 total peptide library of N protein, the cells were collected by centrifugation at 1800 rpm for 5 minutes. Then, 100 μL of Zombie live-death dye was added to each well and the cells were stained in the dark at room temperature for 30 minutes.

[0082] (2) Cell washing: After staining, 150 μL of PBS buffer was added to each well for washing, and the cells were centrifuged again at 1800 rpm for 5 minutes to remove the supernatant.

[0083] (3) Cell surface marker staining: Add 50 μL of PBS solution containing specific fluorescently labeled monoclonal antibodies (targeting cell surface antigens CD3, CD4, and CD8) to the cell pellet, resuspend the cell pellet, and place it in a 4°C refrigerator in the dark for 30 minutes.

[0084] (4) Washing after surface marker staining: After staining, the cells were washed with 150 μL of PBS buffer and centrifuged at 1800 rpm for 5 minutes to remove the supernatant.

[0085] (5) Cell fixation: Add 100 μL of fixative to the cell pellet, resuspend the cell pellet, and place it in a 4°C refrigerator in the dark for 20 minutes.

[0086] (6) Fixative washing, membrane permeabilization, and intracellular factor staining: After fixation, the cells were washed off with 100 μL of 1× permeabilization buffer by centrifugation, and then 200 μL of permeabilization buffer was added to resuspend the cells for permeabilization. The cells were then centrifuged at 1800 rpm for 5 minutes to remove the supernatant. 50 μL of permeabilization buffer containing fluorescently labeled antibodies specific for intracellular factors (IFN-γ, TNF-α, IL-2, IL-4, and IL-10) was added to the cell pellet. After resuspending the cell pellet, the pellet was placed in a 4°C refrigerator in the dark for staining for 30 minutes.

[0087] (7) Washing and sample preparation after intracellular factor staining: After staining, the cells were washed with 150 μL PBS buffer, centrifuged at 1800 rpm for 5 minutes to obtain a cell pellet, and finally resuspended with 200 μL PBS buffer.

[0088] 4. Flow cytometry and data analysis: The prepared cell samples were transferred to flow cytometer tubes and detected using the LSR-Fortessa flow cytometer. The FCS format data were collected and exported using FACSDiva software. The FCS files were then further analyzed using FlowJo X software. For details on the flow gating logic, see Figure 1 .

[0089] Experimental results: Figure 2 As shown, in people who developed breakthrough infection after vaccination with inactivated vaccine, Omicron JN.1 strain N protein-specific CD4 + and CD8 + The levels of IFN-γ, TNF-α and IL-2 secreted by T cells were significantly higher than those of Wuhan-Hu-1 strain N protein-specific T cells. The multifunctional analysis focusing on these three cytokines showed that whether it was Wuhan-Hu-1 or JN.1 strain N protein-specific stimulation, CD4 + and CD8 +There was no statistical difference in the proportion of individuals secreting single, dual or triple cytokines in T cells. Further analysis of the secretion levels of IL-4 and IL-10 cytokines showed that JN.1 strain N protein-specific T cells mainly secreted IFN-γ and TNF-α, while the secretion levels of IL-4 and IL-10 were almost undetectable, showing a typical Th1 and Tc1 phenotype. Overall, breakthrough infection can induce the population to effectively produce N protein-specific memory CD4 cells against the current epidemic strain JN.1. + and CD8 + T cell responses: This population of T cells primarily exhibited Th1 and Tc1 phenotypes and maintained polyfunctionality.

[0090] Example 3

[0091] Detection and analysis of T cell response levels targeting N protein mutation sites and original sites

[0092] 1. Synthesis of small peptide library: Take 10 μL of each of the 14 mutation site peptides of the N protein of the Omicron mutant strain JN.1 substrain and the corresponding 14 original site peptides of the original strain Wuhan-Hu-1 in Table 1, and prepare the N protein mutation site peptide library and original site peptide library respectively.

[0093] 2. Isolation and cryopreservation of PBMCs: Peripheral blood mononuclear cells were extracted from 57 volunteers with breakthrough infection and cryopreserved in liquid nitrogen. The specific steps were the same as the first step in Example 2.

[0094] 3. In vitro stimulation and signal detection using peptide libraries: PBMCs recovered from 57 specimens were stimulated with a library of 14 peptides from the N protein's mutated sites and a library of 14 peptides from the original N protein's original sites. Five hours later, the levels of IFN-γ, TNF-α, and IL-2 secreted by specific T cells were measured by ICS staining and flow cytometry, respectively. For detailed experimental procedures, see Example 2.

[0095] Experimental results: Figure 3 As shown. Figure 3 It can be seen that in people who develop breakthrough infection after vaccination with inactivated vaccine, specific CD4 + The level of IFN-γ secreted by T cells was significantly higher than that of Wuhan-Hu-1 strain N protein-specific T cells, and the specific CD8 + The level of TNF-α secreted by T cells was significantly higher than that of Wuhan-Hu-1 strain N protein-specific T cells. In addition, through correlation analysis, it was found that the mutation site peptide library-specific CD4 + and CD8 +The level of T cell response was significantly correlated with the level of T cell response specific to the original site peptide library. Therefore, we speculate that the JN.1 strain N protein mutant epitope-specific CD4 + and CD8 + The increased T cell response level is due to the emergence of new dominant response epitopes targeting the mutation site, but the specific site remains to be revealed.

[0096] Example 4

[0097] Screening and identification of dominant epitope peptides for the N antigen-specific T cell response of Omicron JN.1 strain

[0098] 1. The 68 overlapping peptides of the Omicron JN.1 strain N antigen (numbered N-1, N-2, N-3, N-4…N-67, N-68) were divided into three-dimensional sub-peptide libraries (Pool 1-Pool 13) according to a three-dimensional matrix. The three-dimensional matrix was constructed by our unit (Ning J, Wang Q, Chen Y, He T, Zhang F, Chen X, et al. Immunodominant SARS-CoV-2-specific-CD4*-and-CD8+-T-cell-responses elicited-by-inactivated-vaccines-in-healthy-adults. J Med Virol 2023; 95(4): e28743.). The specific distribution is as follows:

[0099] (a) The first dimension: includes Pool 1, Pool 2, Pool 3, and Pool 4.

[0100] (b) The second dimension: includes Pool 5, Pool 6, Pool 7, and Pool 8.

[0101] (c) The third dimension: includes Pool 9, Pool 10, Pool 11, Pool 12, and Pool 13.

[0102] The peptides are distributed as follows: each dimension's sub-peptide library contains all 68 peptides from N-1 to N-68. Each peptide appears once in each of the three dimensions' sub-peptide libraries, so it appears three times in total in Pool 1 to Pool 13. For detailed information on the peptides contained in each pool, see Figure 4 .

[0103] 2. Each individual's PBMCs were stimulated in vitro using Pools 1-13, followed by ICS and on-chip testing. The sub-peptide library with the strongest IFN-γ response in each dimension was analyzed. The dominant response epitope peptide for that individual was determined by comparing the shared peptides in the strongest sub-peptide libraries across the three dimensions.

[0104] Experimental results: Figure 5 A and Figure 6 As shown in A, the test results of a representative individual showed that under the stimulation of JN.1 strain N protein, CD4 + and CD8 + The levels of IFN-γ secreted by T cells were significantly increased compared with the negative control. Among the three dimensions of sub-peptide libraries, the sub-peptide libraries with the strongest response levels were Pool 2, Pool 6, and Pool 13. By comparing the peptides contained in these three sub-peptide libraries, it was found that the common peptide was N-38. N-38 was further used to perform a single peptide stimulation experiment on the PBMC of this individual, and the results showed that CD4 + and CD8 + The T cell response level was high, thus verifying that N-38 was the CD4 + and CD8 + T cell immunodominant response peptides.

[0105] 3. In order to observe the dominant frequency of T cell epitope peptides targeting the N protein of the Omicron mutant JN.1 substrain at the population level and identify the most dominant T cell epitope peptide, we used the above-mentioned three-dimensional matrix screening strategy to screen and identify immunodominant epitopes using 57 specimens.

[0106] Experimental results: Figure 5 B and Figure 6 As shown in Figure B, the dominant epitope spectrum of N protein was screened based on 57 samples. The results showed that 9 of the 14 peptide segments covering the mutation sites were dominant response epitopes, namely: N-2, N-4, N-5, N-34, N-37, N-38, N-39, N-67, and N-68.

[0107] Example 5

[0108] Bioinformatics analysis of amino acid conservation of dominant epitopes

[0109] 1. For the immunodominant epitope peptide sequences screened in Example 4, the present invention used MEGA software and Clustal W algorithm to perform sequence homology analysis. After the analysis was completed, the alignment results were exported in FASTA format and imported into ESPript3.0 to generate sequence alignment diagrams (for specific methods, see the literature Kong L, Ma X, Zhang C, Kim SI, Li B, Xie Y, et al. Dual phosphorylation of DGK5-mediated PA burst regulates ROS in plant immunity. Cell. 2024; 187(3): 609-23.e21).

[0110] 2. Sequence data of Omicron substrains (including BA.2.75, XBB, BA.5, BF.7, BA.2.86, and JN.1) were obtained from the 2019 Novel Coronavirus Information Database (https: / / ngdc.cncb.ac.cn / ncov / ).

[0111] Experimental results: Figure 7 As shown, the amino acid sequences of the original strain Wuhan-Hu-1 and six Omicron subvariants were analyzed, and the results showed that epitopes N-2, N-4, N-5, N-34, N-37, N-38, N-39, N-67, and N-68 were almost completely conserved among the six Omicron strains, with a high degree of sequence conservation.

[0112] Example 6

[0113] For the immunodominant epitope peptides screened in Example 4, the present invention used the ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submitfreq_S.php?ran=18132) and AllerTOP v2.1 (https: / / www.ddg-pharmfac.net / allertop_test / ) platforms to perform toxicity and allergenic component analysis, respectively.

[0114] Experimental results: Figure 8 As shown, the results of toxic and allergic component analysis showed that the N-34 and N-67 peptides may contain allergic components and are not suitable for vaccine design, while N-2, N-4, N-5, N-37, N-38, N-39, and N-68 do not contain toxic or allergic components and can be used as candidate antigen epitopes for vaccine design.

[0115] As can be seen from the above examples, the present invention collects PBMCs from breakthrough infected people and synthesizes a full-length peptide library of N proteins covering Wuhan-Hu-1 and JN.1 using a step-by-step method. Through in vitro stimulation combined with intracellular factor staining (ICS) and flow cytometry, it is clear that the memory T cells triggered by breakthrough infection after inactivated vaccine immunization can respond to the N protein of the currently popular Omicron subvariant JN.1, and there is a specific memory T cell response to the mutation site. On this basis, the present invention screens and identifies dominant epitope peptides that can induce T cells to produce a dominant immune response against the mutation site. The dominant epitope peptide can promote the secretion of high levels of antiviral cytokines, thereby enhancing the body's cellular immunity. In addition, the present invention further uses bioinformatics methods to confirm that the screened T cell immunodominant epitopes have a high degree of amino acid sequence conservation in the Omicron mutant strain, and confirms that the T cell immunodominant epitope peptides do not contain toxic components or allergens, thereby effectively reducing the risk of using vaccines prepared based on the epitope peptides. In addition, the epitope peptides provided by the present invention can also be widely used in the research and development of SARS-CoV-2 diagnostic reagents and therapeutic drugs, providing more comprehensive and powerful support and protection against new coronavirus infection.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A T cell immunodominant epitope peptide at a SARS-CoV-2 nucleocapsid protein mutation site, characterized in that: The amino acid sequence of the T cell immunodominant epitope peptide is shown in SEQ ID NO.2 or SEQ ID NOs.4-5 or SEQ ID NOs.10-12 or SEQ ID NO.

14.

2. A nucleic acid molecule encoding a T cell immunodominant epitope peptide at the SARS-CoV-2 nucleocapsid protein mutation site according to claim 1.

3. A T cell immunodominant epitope peptide complex at a SARS-CoV-2 nucleocapsid protein mutation site, characterized in that: The invention comprises the T cell immunodominant epitope peptide according to claim 1.

4. Use of the T cell immunodominant epitope peptide according to claim 1, the nucleic acid molecule according to claim 2, or the T cell immunodominant epitope peptide complex according to claim 3 in the preparation of a new coronavirus drug.

5. Use of the T cell immunodominant epitope peptide according to claim 1, the nucleic acid molecule according to claim 2, or the T cell immunodominant epitope peptide complex according to claim 3 in the preparation of a new coronavirus vaccine.

6. Use of the T cell immunodominant epitope peptide according to claim 1, the nucleic acid molecule according to claim 2, or the T cell immunodominant epitope peptide complex according to claim 3 in the detection of novel coronavirus antigens.

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